Fucosyl transferase gene

ABSTRACT

A DNA molecule is provided which comprises a sequence according to SEQ ID NO: 1 having an open reading frame from base pair 211 to base pair 1740 or having at least 50% homology to the above-indicated sequence, or hybridizing with the above-indicated sequence under stringent conditions, or comprising a sequence which has degenerated to the above-indicated DNA sequence because of the genetic code, the sequence coding for a plant protein having fucosyltransferase activity or being complementary thereto.

This is a continuation of application Ser. No. 09/913,858, filed Aug.20, 2001, pending, which is a 371 application of InternationalApplication No. PCT/AT00/00040, filed Feb. 17, 2000, which claimspriority to A270/99, filed Feb. 18, 1999, all of which are incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to polynucleotides coding for a fucosyltransferase. Furthermore, the invention relates to partial sequences ofthese polynucleotides as well as to vectors comprising thesepolynucleotides, recombinant host cells, plants and insects transfectedwith the polynucleotides or with DNA derived therefrom, respectively, aswell as to glycoproteins produced in these systems.

BACKGROUND OF THE INVENTION

Glycoproteins exhibit a variety and complexity of carbohydrate units,the composition and arrangement of the carbohydrates beingcharacteristic of different organisms. The oligosaccharide units of theglycoproteins have a number of tasks, e.g. they are important inregulating metabolism, they are involved in transmitting cell-cellinteractions, they determine the circulation periods of proteins incirculation, and they are decisive for recognizing epitopes inantigen-antibody reactions.

The glycosylation of glycoproteins starts in the endoplasmatic reticulum(ER), where the oligosaccharides are either bound to asparagine sidechains by N-glycosidic bonds or to serine or threonine side chains byO-glycosidic bonds. The N-bound oligosaccharides contain a common corefrom a penta-saccharide unit which consists of three mannose and twoN-acetyl glucose amine residues. To further modify the carbohydrateunits, the proteins are transported from the ER to the Golgi complex.The structure of the N-bound oligosaccharide units of glycoproteins isdetermined by their conformation and by the composition of the glycosyltransferases of the Golgi compartments in which they are processed.

It has been shown that the core pentasaccharide unit in the Golgicomplex of some plant and insect cells is substituted by xylose andα1,3-bound fucose (P. Lerouge et al., 1998, Plant Mol. Biol. 38, 31-48;Rayon et al., 1998, L. Exp. Bot. 49, 1463-1472). The heptasaccharide“MMXF³” forming constitutes the main oligosaccharide type in plants(Kurosaka et al., 1991, J. Biol. Chem., 266, 4168-4172). Thus, e.g., thehorseradish peroxidase, carrot β-fructosidase and Erythrina cristagallicomprise lectin as well as the honeybee venom phospholipase A2 or theneuronal membrane glycoproteins from insect embryos α1,3-fucose residueswhich are bound to the glycan core. These structures are also termedcomplex N-glycans or mannose-deficient or truncated N-glycans,respectively. The α-mannosyl residues may be further replaced by GlcNAc,to which galactose and fucose are bound so that a structure is preparedwhich corresponds to the human Lewis a-epitope (Melo et al., 1997, FEBSLett 415, 186-191; Fitchette-Laine et al., 1997, Plant J. 12,1411-1417).

Neither xylose nor the α1,3-bound fucose exist in mammalianglycoproteins. It has been found that the core-α1,3-fucose plays animportant role in the epitope recognition of antibodies which aredirected against plant and insect N-bound oligosaccharides (I. B. H.Wilson et al., Glycobiology Vol. 8, No. 7, pp. 651-661, 1998), andthereby trigger immune reactions in human or animal bodies against theseoligosaccharides. The α1,3-fucose residue furthermore seems to be one ofthe main causes for the wide-spread allergic cross reactivity betweenvarious plant and insect allergens (Tretter et al., Int. Arch. AllergyImmunol. 1993; 102:259-266) and is also termed “cross-reactivecarbohydrate determinant” (CCD). In a study of epitopes of tomatoes andgrass pollen, also α1,3-bound fucose residues were found as a commondeterminant, which seems to be the reason why tomato and grass pollenallergies frequently occur together in patients (Petersen et al., 1996,J. Allergy Clin. Immunol., Vol. 98, 4; 805-814). Due to the frequentoccurrence of immunological cross reactions, the CCDs moreover maskallergy diagnoses.

The immunological reactions triggered in the human body by plantproteins are the main problem in the medicinal use of recombinant humanproteins produced in plants. To circumvent this problem,α1,3-core-fucosylation would have to be prevented. In a study it couldbe demonstrated that oligosaccharides comprising an L-galactose insteadof an L-fucose (6-deoxy-L-galactose) nevertheless are biologically fullyactive (E. Zablackis et al., 1996, Science, Vol. 272). According toanother study, a mutant of the plant Arabidopsis thaliana was isolatedin which the N-acetyl-glucosaminyl transferase I, the first enzyme inthe biosynthesis of complex glycans, is missing. The biosynthesis of thecomplex glycoproteins in this mutant thus is disturbed. Nevertheless,these mutant plants are capable of developing normally under certainconditions (A. Schaewen et al, 1993, Plant Physiol. 102; 1109-1118).

To purposefully block the binding of the core-α1,3-fucose in anoligosaccharide without also interfering in other glycosylation steps,merely that enzyme would have to be inactivated which is directlyresponsible for this specific glycosylation, i.e. the core-α1,3-fucosyltransferase. It has been isolated and characterized for the first timefrom mung beans, and it has been found that the activity of this enzymedepends on the presence of non-reducing GlcNAc ends (Staudacher et al.,1995, Glycoconjugate J. 12, 780-786). This transferase which only occursin plants and insect, yet not in human beings or in other vertebrates,would have to be inactivated on purpose or suppressed so that humanproteins which are produced in plants or in plant cells or also ininsects or in insect cells, respectively, do no longer comprise thisimmune-reaction-triggering epitope, as has been the case so far.

The publication by John M. Burke “Clearing the way for ribozymes”(Nature Biotechnology 15:414-415; 1997) relates to the general mode offunction of ribozymes.

The publication by Pooga et al., “Cell penetrating PNA constructsregulate galanin receptor levels and modify pain transmission in vivo”(Nature Biotechnology 16:857-861; 1998) relates to PNA molecules ingeneral and specifically to a PNA molecule that is complementary tohuman galanin receptor type 1 mRNA.

U.S. Pat. No. 5,272,066 A relates to a method of changing eukaryotic andprokaryotic proteins to prolongue their circulation in vivo. In thisinstance, the bound oligosaccharides are changed with the help ofvarious enzymes, among them also GlcNAc-α1→3(4)-fucosyl transferase.

EP 0 643 132 A1 relates to the cloning of an α1,3-fucosyl transferaseisolated from human cells (THP-1). The carbohydrate chains described inthis publication correspond to human sialyl Lewis x- and sialyl Lewisa-oligosaccharides. The specificity of the enzyme from human cells isquite different than that of fucosyltransferase from plant cells.

DESCRIPTION OF THE INVENTION

It is an object of the present invention to clone and to sequence thegene which codes for a plant fucosyl transferase, and to prepare vectorscomprising this gene, DNA fragments thereof or an altered DNA or a DNAderived therefrom, to transfect plants and insects as well as cellsthereof with one of these vectors, to produce glycoproteins that do notcomprise the normally occurring α1,3-core-fucose, as well as to providecorresponding methods therefor.

The object according to the invention is achieved by a DNA moleculecomprising a sequence according to SEQ ID NO: 1 (in this disclosure alsothe IUPAC code has been used, “N” meaning inosin) with an open readingframe from base pair 211 to base pair 1740 or being at least 50%homologous to the above sequence or hybridizing with the above-indicatedsequence under stringent conditions, or comprising a sequence which hasdegenerated to the above DNA sequence due to the genetic code, thesequence coding for a plant protein which has fucosyl transferaseactivity or is complementary thereto.

This sequence which has not been described before can be perfectly usedfor any experiments, analysis and methods for production etc. whichrelate to the plant fucosyl transferase activity. Here the DNA sequenceas well as the protein coded by this sequence are of interest. However,in particular the DNA sequence will be used for the inhibition of thefucosyl transferase activity.

The open reading frame of the SEQ ID NO: 1 codes for a protein with 510amino acids and with a theoretical molecular weight of 56.8 kDa, atransmembrane portion presumably being present in the region betweenAsn36 and Gly54. The calculated pI value of the encoded protein of thesequence according to SEQ ID NO: 1 is 7.51.

The activity of the plant fucosyl transferase is detected by a methodand measured, the fucosyl transferase being added to a sample comprisinglabelled fucose and an acceptor (e.g. a glycoprotein) bound to acarrier, e.g. Sepharose. After the reaction time, the sample is washed,and the content of bound fucose is measured. The activity of the fucosyltransferase in this case is seen as positive if the activity measurementis higher by at least 10 to 20%, in particular at least 30 to 50%, thanthe activity measurement of the negative control. The structure of theglycoprotein may additionally be verified by means of HPLC. Suchprotocols are prior art (Staudacher et al. 1998, Anal. Biochem. 246,96-101; Staudacher et al. 1991, Eur. J. Biochem. 199, 745-751).

For example, fucosyl transferase is admixed to a sample comprisingradioactively labelled fucose and an acceptor, e.g.GlcNAcβ1-2Manα1-3(GlcNAβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn. Afterthe reaction time, the sample is purified by anion exchangechromatography, and the content of bound fucose is measured. From thedifference of the measured radioactivity of the sample with acceptor andthat of a negative control without acceptor, the activity can becalculated. The activity of the fucosyl transferase is already evaluatedas positive if the radioactivity measured is at least 30-40% higher thanthe measured radioactivity of the negative sample.

The pairing of two DNA molecules can be changed by selection of thetemperature and ionic strength of the sample. By stringent conditions,according to the invention conditions are understood which allow for anexact, stringent, binding. For instance, the DNA molecules arehybridized in 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4, pH 7.0, 1 mMEDTA at 50° C., and washed with 1% SDS at 42° C.

Whether sequences have an at least 50% homology to SEQ ID NO: 1 can bedetermined e.g. by means of the program FastDB of EMBL or SWISSPROT databank.

Preferably, the sequence of the DNA molecule of the invention encodes aprotein with a GlcNAc-α1,3-fucosyl transferase activity, in particularwith a core-α1,3-fucosyl transferase activity.

As described above the core of α1,3-fucosyl transferase is present inplants and insects, however, not in the human body, so that inparticular this DNA sequence is useful in analysis and experiments aswell as methods for production which are fucosyl transferase specific.

By a core-α1,3-fucosyl transferase, in particular GDP-L-Fuc:Asn-boundGlcNAc-α1,3-fucosyl transferase is understood. Within the scope of thepresent invention, the term α1,3-fucosyl transferase as a ruleparticularly means core-α1,3 fucosyl transferase. For theabove-described activity measurement, in particular acceptors having anon-reducing GlcNAc terminus are used. Such acceptors are, e.g.,GlcNAcβ1-2Manα1-3(GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAβ1-Asn,GlcNAcβ1-2Manα1-3(GlcNAcβ1-2Manα1-6)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAcβ1-AsnandGlcNAcβ1-2Manα1-3[Manα1-3(Manα1-6)Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn.Whether the fucose is bound or not can furthermore be determined bymeasuring the insensitivity relative to N-glycosidase F, which can bedetected by means of mass spectrometry.

Preferably, the DNA molecule according to the invention comprises atleast 70-80%, particularly preferred at least 95%, homology to thesequence according to SEQ ID NO: 1. This sequence codes for aparticularly active GlcNAc-α1,3-fucosyl transferase.

Since the DNA sequence can be more or less changed according to theplant or the insect a sequence which shows, for example, 70% homology toa sequence according to SEQ ID No 1 has also a fucosyl transferaseactivity which is sufficient in order to be used in analysis,experiments or methods of production as above described.

According to a further advantageous embodiment, the DNA moleculecomprises 2150 to 2250, in particular 2198, base pairs. This DNAmolecule comprises 100 to 300, preferably 210, base pairs upstream infront of the start codon, as well as 350 to 440, in particular 458, basepairs downstream after the stop codon of the open reading frame, whereinthe end of the DNA molecule preferably comprises a 3′-poly(A)-tail. Inthis manner, a faultless regulation on translation level is ensured anda DNA molecule is provided which is particularly efficient andunproblematic for the coding of an active GlcNAc-α1,3-fucosyltransferase.

The present invention moreover relates to a DNA molecule which comprisesa sequence according to SEQ ID NO: 3 or comprising a sequence having atleast 85%, particularly preferred at least 95%, in particular at least99%, homology to the above-identified sequence or which, under stringentconditions, hybridizes with the above-indicated sequence or which hasdegenerated to the above-indicated DNA sequence due to the genetic code.The homology preferably is determined with a program which recognizesinsertions and deletions and which does not consider these in thehomology calculation. This nucleotide sequence codes for a conservedpeptide motif, which means that the plurality of the active andfunctioning GlcNAc-α1,3-fucosyl transferases comprises the amino acidsequence encoded thereby. In this instance, the sequence may either havethe same size as the sequence according to SEQ ID NO: 3, or, of course,it may also be larger. This sequence has a smaller length than thesequence which codes the complete protein and is therefore lesssensitive with respect to recombination, deletion, or any othermutations. Due to the conservative motif and its higher stability thissequence is particularly advantageous for sequence recognising test.

SEQ ID NO: 3 comprises the following sequence:5′-GAAGCCCTGAAGCACTACAAATTTAGCTTAGCGTTTGAAAATTCGAATGAGGAAGATTATGTAACTGAAAAATTCTTCCAATCCCTTGTTGCTGGAA CTGTCCCT-3′

In a further aspect, the present invention relates to a DNA moleculewhich comprises a partial sequence of one of the above-indicated DNAmolecules and has a size of from 20 to 200, preferably from 30 to 50,base pairs. The DNA molecule may, e.g., be utilized to bind, as a probe,to complementary sequences of GlcNAc-α1,3-fucosyl transferases so thatthey can be selected from a sample. In this manner, furtherGlcNAc-α1,3-fucosyl transferases from the most varying plants andinsects can be selected, isolated and characterized. Any desired one oralso several different partial sequences may be used, in particular apart of the conserved motif already described above.

In doing so, it is particularly advantageous if one of theabove-indicated DNA molecules is covalently associated with a detectablelabelling substance. As the labelling substance, any common marker canbe used, such as, e.g., fluorescent, luminescent, radioactive markers,non-isotopic markers, such as biotin, etc. In this manner, reagents areprovided which are suitable for the detection, selection andquantitation of corresponding DNA molecules in solid tissue samples(e.g. from plants) or also in liquid samples, by means of hybridizingmethods.

A further aspect of the invention relates to a biologically functionalvector which comprises one of the above-indicated DNA molecules or partsthereof of differing lengths with at least 20 base pairs. Fortransfection into host cells, an independent vector capable ofamplification is necessary, wherein, depending on the host cell,transfection mechanism, task and size of the DNA molecule, a suitablevector can be used. Since a large number of different vectors is known,an enumeration thereof would go beyond the limits of the presentapplication and therefore is done without here, particularly since thevectors are very well known to the skilled artisan (as regards thevectors as well as all the techniques and terms used in thisspecification which are known to the skilled artisan, cf. also SambrookManiatis). Ideally, the vector has a small molecule mass and shouldcomprise selectable genes so as to lead to an easily recognizablephenotype in a cell so thus enable an easy selection ofvector-containing and vector-free host cells. To obtain a high yield ofDNA and corresponding gene products, the vector should comprise a strongpromoter, as well as an enhancer, gene amplification signals andregulator sequences. For an autonomous replication of the vector,furthermore, a replication origin is important. Polyadenylation sitesare responsible for correct processing of the mRNA and splice signalsfor the RNA transcripts. If phages, viruses or virus particles are usedas the vectors, packaging signals will control the packaging of thevector DNA. For instance, for transcription in plants, Ti plasmids aresuitable, and for transcription in insect cells, baculoviruses, and ininsects, respectively, transposons, such as the P element.

If the above-described inventive vector is inserted into a plant or intoa plant cell, a post-transcriptional suppression of the gene expressionof the endogenous α1,3-fucosyl transferase gene is attained bytranscription of a transgene homologous thereto or of parts thereof, insense orientation. For this sense technique, furthermore, reference ismade to the publications by Baucombe 1996, Plant. Mol. Biol., 9:373-382,and Brigneti et al., 1998, EMBO J. 17:6739-6746. This strategy of “genesilencing” is an effective way of suppressing the expression of theα1,3-fucosyl transferase gene, cf. also Waterhouse et al., 1998, Proc.Natl. Acad. Sci. USA, 95:13959-13964.

Furthermore, the invention relates to a biologically functional vectorcomprising a DNA molecule according to one of the above-describedembodiments, or parts thereof of differing lengths in reverseorientation to the promoter. If this vector is transfected in a hostcell, an “antisense mRNA” will be read which is complementary to themRNA of the GlcNAc-α1,3-fucosyl transferase and complexes the latter.This bond will either hinder correct processing, transportation,stability or, by preventing ribosome annealing, it will hindertranslation and thus the normal gene expression of theGlcNAc-α1,3-fucosyl transferase.

Although the entire sequence of the DNA molecule could be inserted intothe vector, partial sequences thereof because of their smaller size maybe advantageous for certain purposes. With the antisense aspect, e.g.,it is important that the DNA molecule is large enough to form asufficiently large antisense MRNA which will bind to the transferasemRNA. A suitable antisense RNA molecule comprises, e.g., from 50 to 200nucleotides since many of the known, naturally occurring antisense RNAmolecules comprise approximately 100 nucleotides.

For a particularly effective inhibition of the expression of an activeα1,3-fucosyl transferase, a combination of the sense technique and theantisense technique is suitable (Waterhouse et al., 1998, Proc. Natl.Acad. Sci., USA, 95:13959-13964).

Advantageously, rapidly hybridizing RNA molecules are used. Theefficiency of antisense RNA molecules which have a size of more than 50nucleotides will depend on the annealing kinetics in vitro. Thus, e.g.,rapidly annealing antisense RNA molecules exhibit a greater inhibitionof protein expression than slowly hybridizing RNA molecules (Wagner etal., 1994, Annu. Rev. Microbiol., 48:713-742; Rittner et al., 1993,Nucl. Acids Res., 21:1381-1387). Such rapidly hybridizing antisense RNAmolecules particularly comprise a large number of external bases (freeends and connecting sequences), a large number of structural subdomains(components) as well as a low degree of loops (Patzel et al. 1998;Nature Biotechnology, 16; 64-68). The hypothetical secondary structuresof the antisense RNA molecule may, e.g., be determined by aid of acomputer program, according to which a suitable antisense RNA DNAsequence is chosen.

Different sequence regions of the DNA molecule may be inserted into thevector. One possibility consists, e.g., in inserting into the vectoronly that part which is responsible for ribosome annealing. Blocking inthis region of the mRNA will suffice to stop the entire translation. Aparticularly high efficiency of the antisense molecules also results forthe 5′- and 3′-nontranslated regions of the gene.

Preferably, the DNA molecule according to the invention includes asequence which comprises a deletion, insertion and/or substitutionmutation. The number of mutant nucleotides is variable and varies from asingle one to several deleted, inserted or substituted nucleotides. Itis also possible that the reading frame is shifted by the mutation. Insuch a “knock-out gene” it is merely important that the expression of aGlcNAc-α1,3-fucosyl transferase is disturbed, and the formation of anactive, functional enzyme is prevented. In doing so, the site of themutation is variable, as long as expression of an enzymatically activeprotein is prevented. Preferably, the mutation in the catalytic regionof the enzyme which is located in the C-terminal region. The method ofinserting mutations in DNA sequences are well known to the skilledartisan, and therefore the various possibilities of mutageneses need notbe discussed here in detail. Coincidental mutageneses as well as, inparticular, directed mutageneses, e.g. the site-directed mutagenesis,oligonucleotide-controlled mutagenesis or mutageneses by aid ofrestriction enzymes may be employed in this instance.

The invention further provides a DNA molecule which codes for a ribozymewhich comprises two sequence portions of at least 10 to 15 base pairseach, which are complementary to sequence portions of an inventive DNAmolecule as described above so that the ribozyme complexes and cleavesthe mRNA which is transcribed from a natural GlcNAc-α1,3-fucosyltransferase DNA molecule. The ribozyme will recognized the MRNA of theGlcNAc-α1,3-fucosyl transferase by complementary base pairing with themRNA. Subsequently, the ribozyme will cleave and destroy the RNA in asequence-specific manner, before the enzyme is translated. Afterdissociation from the cleaved substrate, the ribozyme will repeatedlyhybridize with RNA molecules and act as specific endonuclease. Ingeneral, ribozymes may specifically be produced for inactivation of acertain mRNA, even if not the entire DNA sequence which codes for theprotein is known. Ribozymes are particularly efficient if the ribosomesmove slowly along the mRNA. In that case it is easier for the ribozymeto find a ribosome-free site on the mRNA. For this reason, slow ribosomemutants are also suitable as a system for ribozymes (J. Burke, 1997,Nature Biotechnology; 15, 414-415). This DNA molecule is particularlyadvantageous for the downregulation and inhibition, respectively, of theexpression of plant GlcNAc-α1,3-fucosyl transferases.

One possible way is also to use a varied form of a ribozmye, i.e. aminizyme. Minizymes are efficient particularly for cleaving larger mRNAmolecules. A minizyme is a hammer head ribozyme which has a shortoligonucleotide linker instead of the stem/loop II. Dimer-minizymes areparticularly efficient (Kuwabara et al., 1998, Nature Biotechnology, 16;961-965).

Consequently, the invention also relates to a biologically functionalvector which comprises one of the two last-mentioned DNA molecules(mutation or ribozyme-DNA molecule). What has been said above regardingvectors also applies in this instance. Such a vector can be, forexample, inserted into a microorganism and can be used for theproduction of high concentrations of the above described DNA molecules.Furthermore such a vector is particularly good for the insertion of aspecific DNA molecule into a plant or an insect organism in order todownregulate or completely inhibit the GlcNAc-α1,3-fucosyl transferaseproduction in this organism.

According to the invention, there is provided a method of preparing acDNA comprising the DNA molecule of the invention, wherein RNA isisolted from an insect or plant cell, in particular from hypokotylcells, by means of which a reverse transcription is carried out afterhaving admixed a reverse transcriptase and primers. The individual stepsof this method are carried out according to protocols known per se. Forthe reverse transcription, on the one hand, it is possible to producethe cDNA of the entire mRNA with the help of oligo(dT) primers, and onlythen to carry out a PCR by means of selected primers so as to prepareDNA molecules comprising the GlcNAc-α1,3-fucosyl transferase gene. Onthe other hand, the selected primers may directly be used for thereverse transcription so as to obtain short, specific cDNA. The suitableprimers may be prepared e.g. synthetically according to the pattern ofcDNA sequences of the transferase. With the help of this method bigquantities of the inventive cDNA molecules can be produced quickly in asimple way and with few mistakes.

The invention furthermore relates to a method of cloning aGlcNAc-α1,3-fucosyl transferase, characterized in that the DNA moleculeof the invention is cloned into a vector which subsequently istransfected into a host cell or host, respectively, wherein, byselection and amplification of transfected host cells, cell lines areobtained which express the active GlcNac-α1,3-fucosyl transferase. TheDNA molecule is inserted into the vector by aid of restrictionendonucleases, e.g. For the vector, there applies what has already beensaid above. What is important in this method is that an efficienthost-vector system is chosen. To obtain an active enzyme, eukaryotichost cells are particularly suitable. One possible way is to transfectthe vector in insect cells. In doing so, in particular an insect viruswould have to be used as vector, such as, e.g., baculovirus.

Of course, human or other vertebrate cells can also be transfected, inwhich case the latter would express an enzyme foreign to them.

Preferably, a method of preparing recombinant host cells, in particularplant or insect cells, or plants or insects, respectively, with asuppressed or completely stopped GlcNac-α1,3-fucosyl transferaseproduction is provided, which is characterized in that at least one ofthe vectors according to the invention, i.e. that one comprising theinventive DNA molecule, the mutant DNA molecule or the DNA moleculecoding for ribozymes or the one comprising the DNA molecule in inverseorientation to the promoter, is inserted into the host cell or plant orinto the insect. What has been said above for the transfection also isapplicable in this case.

As the host cells, plant cells may, e.g., be used, wherein, e.g., the Tiplasmid with the agrobacterium system is eligible. With theagrobacterium system it is possible to transfect a plant directly:agrobacteria cause root stem galls inplants. If agrobacteria infect aninjured plant, the bacteria themselves do not get into the plant, butthey insert the recombinant DNA portion, the so-called T-DNA, from theannular, extra chromosomal, tumour-inducing Ti-plasmid into the plantcells. The T-DNA, and thus also the DNA molecule inserted therein, areinstalled in the chromosomal DNA of the cell in a stable manner so thatthe genes of the T-DNA will be expressed in the plant.

There exist numerous known, efficient transfection mechanisms fordifferent host systems. Some examples are electroporation, the calciumphosphate method, microinjection, liposome method.

Subsequently, the transfected cells are selected, e.g. on the basis ofantibiotic resistences for which the vector comprises genes, or othermarker genes. Then the transfected cell lines are amplified, either insmall amounts, e.g. in Petri dishes, or in large amounts, e.g. infermentors. Furthermore, plants have a particular characteristic, i.e.they are capable to re-develop from one (transfected) cell or from aprotoplast, respectively, to a complete plant which can be grown.

Depending on the vector used, processes will occur in the host so thatthe enzyme expression will be suppressed or completely blocked:

If the vector comprising the DNA molecule with the deletion, insertionor substitution mutation is transfected, a homologous recombination willoccur: the mutant DNA molecule will recognize the identical sequence inthe genome of the host cell despite its mutation and will be insertedexactly on that place so that a “knock-out gene” is formed. In thismanner, a mutation is introduced into the gene for theGlcNAc-α1,3-fucosyl transferase which is capable of inhibiting thefaultless expression of the GlcNAc-α1,3-fucosyl transferase. As has beenexplained above, with this technique it is important that the mutationsuffices to block the expression of the active protein. After selectionand amplification, the gene may be sequenced as an additional check soas to determine the success of the homologous recombination or thedegree of mutation, respectively.

If the vector comprising the DNA molecule coding for a ribozyme istransfected, the active ribozyme will be expressed in the host cell. Theribozyme complexes the complementary mRNA sequence of theGlcNAc-α1,3-fucosyl transferase at least at a certain site, cleaves thissite, and in this manner it can inhibit the translation of the enzyme.In this host cell as well as in cell lines, or optionally, plant,respectively, derived therefrom, GlcNAc-α1,3-fucosyl transferase willnot be expressed.

In case the vector comprises the inventive DNA molecule in sense orinverse direction to the promoter, a sense or antisense-mRNA will beexpressed in the transfected cell (or plant, respectively). Theantisense mRNA is complementary at least to a part of the mRNA sequenceof the GlcNAc-α1,3-fucosyl transferase and may likewise inhibittranslation of the enzyme. As an example of a method of suppressing theexpression of a gene by antisense technique, reference is made to thepublication by Smith et al., 1990, Mol. Gen. Genet. 224:477-481, whereinin this publication the expression of a gene involved in the maturingprocess of tomatoes is inhibited.

In all the systems, expression of the GlcNAc-α1,3-fucosyl transferase isat least suppressed, preferably even completely blocked. The degree ofthe disturbance of the gene expression will depend on the degree ofcomplexing, homologous recombination, on possible subsequentcoincidental mutations and on other processes in the region of thegenome. The transfected cells are checked for GlcNac-α1,3-fucosyltransferase activity and selected.

Moreover, it is possible to still further increase the above-describedsuppression of the expression of the α1,3-fucosyl transferase byintroducing into the host a vector comprising a gene coding for amammalian protein, e.g. β1,4-galactosyl transferase, in addition to theinsertion of an above-described vector. Fucosylation may be reduced bythe action of other mammalian enzymes, the combination of the inhibitionof the expression of an active α1,3-fucosyl transferase by means of theinventive vector and by means of a mammalian enzyme vector beingparticularly efficient.

Any type of plant may be used for transfection, e.g. mung bean, tobaccoplant, tomato and/or potato plant.

Another advantageous method of producing recombinant host cells, inparticular plant or insect cells, or plants or insects, respectively,consists in that the DNA molecule comprising the mutation is insertedinto the genome of the host cell, or plant or insect, respectively, inthe place of the non-mutant homologous sequence (Schaefer et al., 1997,Plant J.; 11(6):1195-1206). This method thus does not function with avector, but with a pure DNA molecule. The DNA molecule is inserted intothe host e.g. by gene bombardment, microinjection or electroporation, tomention just three examples. As has already been explained, the DNAmolecule binds to the homologous sequence in the genome of the host sothat a homologous recombination and thus reception of the deletion,insertion or substitution mutation, respectively, will result in thegenome: Expression of the GlcNAc-α1,3-fucosyl transferase can besuppressed or completely blocked, respectively.

A further aspect of the invention relates to plants or plant cells,respectively, as well as insect or insect cells, respectively, theirGlcNAc-α1,3-fucosyl transferase activity being less than 50%, inparticular less than 20%, particularly preferred 0%, of theGlcNAc-α1,3-fucosyl transferase activity occurring in natural plants orplant cells, respectively, and insects or insect cells, respectively.The advantage of these plants or plant cells, respectively, is that theglycoproteins produced by them do not comprise any or hardly compriseany α1,3-bound fucose. If products of these plants or insects,respectively, are taken up by human or vertebrate bodies, there will beno immune reaction to the α1,3-fucose epitope.

Preferably, recombinant plants or plant cells, respectively, areprovided which have been prepared by one of the methods described above,their GlcNAc-α1,3-fucosyl transferase production being suppressed orcompletely blocked, respectively.

The invention also relates to recombinant insects or insect cells,respectively, which have been prepared by one of the methods describedabove and whose GlcNAc-α1,3-fucosyl transferase production is suppressedor completely blocked, respectively. Also in this instance, noglycoproteins having α1,3-bound fucose residues are produced so thatlikewise no immune reaction to the α1,3-fucose epitope will occur.

The invention also relates to a PNA molecule comprising a base sequencecomplementary to the sequence of the DNA molecule according to theinvention as well as partial sequences thereof. PNA (peptide nucleicacid) is a DNA-like sequence, the nucleobases being bound to apseudo-peptide backbone. PNA generally hybridizes with complementaryDNA-, RNA- or PNA-oligomers by Watson-Crick base pairing and helixformation. The peptide backbone ensures a greater resistance toenzymatic degradation. The PNA molecule thus is an improved antisenseagent. Neither nucleases nor proteases are capable of attacking a PNAmolecule. The stability of the PNA molecule, if bound to a complementarysequence, comprises a sufficient steric blocking of DNA and RNApolymerases, reverse transcriptase, telomerase and ribosomes.

If the PNA molecule comprises the above-mentioned sequence, it will bindto the DNA or to a site of the DNA, respectively, which codes forGlcNAc-α1,3-fucosyl transferase and in this way is capable of inhibitingtranscription of this enzyme. As it is neither transcribed nortranslated, the PNA molecule will be prepared synthetically, e.g. by aidof the the t-Boc technique.

Advantageously, a PNA molecule is provided which comprises a basesequence which corresponds to the sequence of the inventive DNA moleculeas well as partial sequences thereof. This PNA molecule will complex themRNA or a site of the mRNA of GlcNAc-α1,2-fucosyl transferase so thatthe translation of the enzyme will be inhibited. Similar arguments asset forth for the antisense RNA apply in this case. Thus, e.g., aparticularly efficient complexing region is the translation start regionor also the 5′-non-translated regions of mRNA.

A further aspect of the present invention relates to a method ofpreparing plants or insects, or cells, respectively, in particular plantor insect cells which comprise a blocked expression of theGlcNAc-α1,3-fucosyl transferase on transcription or translation level,respectively, which is characterized in that inventive PNA molecules areinserted in the cells. To insert the PNA molecule or the PNA molecules,respectively, in the cell, again conventional methods, such as, e.g.,electroporation or microinjection, are used. Particularly efficient isinsertion if the PNA oligomers are bound to cell penetration peptides,e.g. transportan or pAntp (Pooga et al., 1998, Nature Biotechnology, 16;857-861).

The invention provides a method of preparing recombinant glycoproteinswhich is characterized in that the inventive, recombinant plants orplant cells, respectively, as well as recombinant insects or insectcells, respectively, whose GlcNAc-α1,3-fucosyl transferase production issuppressed or completely blocked, respectively, or plants or insects, orcells, respectively, in which the PNA molecules have been insertedaccording to the method of the invention, are transfected with the genethat expresses the glycoprotein so that the recombinant glycoproteinsare expressed. In doing so, as has already been described above, vectorscomprising genes for the desired proteins are transfected into the hostor host cells, respectively, as has also already been described above.The transfected plant or insect cells will express the desired proteins,and they have no or hardly any α1,3-bound fucose. Thus, they do nottrigger the immune reactions already mentioned above in the human orvertebrate body. Any proteins may be produced in these systems.

Advantageously, a method of preparing recombinant human glycoproteins isprovided which is characterized in that the recombinant plants or plantcells, respectively, as well as recombinant insects or insect cells,respectively, whose GlcNAc-α1,3-fucosyl transferase production issuppressed or completely blocked, or plants or insects, or cells,respectively, in which PNA molecules have been inserted according to themethod of the invention, are transfected with the gene that expressesthe glycoprotein so that the recombinant glycoproteins are expressed. Bythis method it becomes possible to produce human proteins in plants(plant cells) which, if taken up by the human body, do not trigger anyimmune reaction directed against α1,3-bound fucose residues. There, itis possible to utilize plant types for producing the recombinantglycoproteins which serve as food stuffs, e.g. banana, potato and/ortomato. The tissues of this plant comprise the recombinant glycoproteinso that, e.g. by extraction of the recombinant glycoprotein from thetissue and subsequent administration, or directly by eating the planttissue, respectively, the recombinant glycoprotein is taken up in thehuman body.

Preferably, a method of preparing recombinant human glycoproteins formedical use is provided, wherein the inventive, recombinant plants orplant cells, respectively, as well as recombinant insects or insectcells, respectively, whose GlcNAc-α1,3-fucosyl transferase production issuppressed or completely blocked, respectively, or plants or insects, orcells, respectively, into which the PNA molecules have been insertedaccording to the method of the invention, are transfected with the genethat expresses the glycoprotein so that the recombinant glycoproteinsare expressed. In doing so, any protein can be used which is of medicalinterest.

Moreover, the present invention relates to recombinant glycoproteinsaccording to a method described above, wherein they have been preparedin plant or insect systems and wherein their peptide sequence comprisesless than 50%, in particular less than 20%, particularly preferred 0%,of the α1,3-bound fucose residues occurring in proteins expressed innon-fucosyl transferase-reduced plant or insect systems. Naturally,glycoproteins which do not comprise α1,3-bound fucose residues are to bepreferred. The amount of α1,3-bound fucose will depend on the degree ofthe above-described suppression of the GlcNAc-α1,3-fucosyl transferase.

Preferably, the invention relates to recombinant human glycoproteinswhich have been produced in plant or insect systems according to amethod described above and whose peptide sequence comprises less than50%, in particular less than 20%, particularly preferred 0%, of theα1,3-bound fucose residues occurring in the proteins expressed innon-fucosyl transferase-reduced plant or insect systems.

A particularly preferred embodiment relates to recombinant humanglycoproteins for medical use which have been prepared in plant orinsect systems according to a method described above and whose peptidesequence comprises less than 50%, in particular less than 20%,particularly preferred 0%, of the α1,3-bound fucose residues occurringin the proteins expressed in non-fucosyl transferase-reduced plant orinsect systems.

The glycoproteins according to the invention may include other boundoligosaccharide units specific for plants or insects, respectively,whereby—in the case of human glycoproteins—they differ from thesenatural glycoproteins. Nevertheless, by the glycoproteins according tothe invention, a slighter immune reaction or no immune reaction at all,respectively, is triggered in the human body, since, as has already beenexplained in the introductory portion of the specification, theα1,3-bound fucose residues are the main cause for the immune reactionsor cross immune reaction, respectively, to plant and insectglycoproteins.

A further aspect comprises a pharmaceutical composition comprising theglycoproteins according to the invention. In addition to theglycoproteins of the invention, the pharmaceutical composition comprisesfurther additions common for such compositions. These are, e.g.,suitable diluting agents of various buffer contents (e.g. Tris-HCl,acetate, phosphate, pH and ionic strength, additives, such as tensidesand solubilizers (e.g. Tween 80, Polysorbate 80), preservatives (e.g.Thimerosal, benzyl alcohol), adjuvants, antioxidants (e.g. ascorbicacid, sodium metabisulfite), emulsifiers, fillers (e.g. lactose,mannitol), covalent bonds of polymers, such as polyethylene glycol, tothe protein, incorporation of the material in particulate compositionsof polymeric compounds, such as polylactic acid, polyglycolic acid, etc.or in liposomes, auxiliary agents and/or carrier substances which aresuitable in the respective treatment. Such compositions will influencethe physical condition, stability, rate of in vivo liberation and rateof in vivo excretion of the glycoproteins of the invention.

The invention also provides a method of selecting DNA molecules whichcode for a GlcNAc-α1,3-fucosyl transferase, in a sample, wherein thelabelled DNA molecules of the invention are admixed to the sample, whichbind to the DNA molecules that code for a GlcNAc-α1,3-fucosyltransferase. The hybridized DNA molecules can be detected, quantitatedand selected. For the sample to contain single strand DNA with which thelabelled DNA molecules can hybridize, the sample is denatured, e.g. byheating.

One possible way is to separate the DNA to be assayed, possibly afterthe addition of endonucleases, by gele electrophoresis on an agarosegel. After having been transferred to a membrane of nitrocellulose, thelabelled DNA molecules according to the invention are admixed whichhybridize to the corresponding homologous DNA molecule (“Southernblotting”).

Another possible way consists in finding homologous genes from otherspecies by PCR-dependent methods using specific and/or degeneratedprimers, derived from the sequence of the DNA molecule according to theinvention.

Preferably, the sample for the above-identified inventive methodcomprises genomic DNA of a plant or insect organism. By this method, alarge number of plants and insects is assayed in a very rapid andefficient manner for the presence of the GlcNAc-α1,3-fucosyl transferasegene. In this manner, it is respectively possible to select plants andinsects which do not comprise this gene, or to suppress or completelyblock, respectively, the expression of the GlcNAc-α1,3-fucosyltransferase in such plants and insects which comprise this gene, by anabove-described method of the invention, so that subsequently they maybe used for the transfection and production of (human) glycoproteins.

The invention also relates to DNA molecules which code for aGlcNAc-α1,3-fucosyl transferase which have been selected according tothe two last-mentioned methods and subsequently have been isolated fromthe sample. These molecules can be used for further assays. They can besequenced and in turn can be used as DNA probes for findingGlcNAc-α1,3-fucosyl transferases. These—labelled—DNA molecules willfunction for organisms, which are related to the organisms from whichthey have been isolated, more efficiently as probes than the DNAmolecules of the invention.

A further aspect of the invention relates to a preparation ofGlcNAc-α1,3-fucosyl transferase cloned according to the invention whichcomprises isoforms having pI values of between 6.0 and 9.0, inparticular between 6.8 and 8.2. The pI values of a protein is that pHvalue at which its net charge is zero and is dependent on the amino acidsequence, the glycosylation pattern as well as on the spatial structureof the protein. The GlcNAc-α1,3-fucosyl transferase comprises at least 7isoforms which have a pI value in this range. The reason for the variousisoforms of the transferase are, e.g., different glycosylations as wellas limited proteolysis. Tests have shown that mung bean seedlings ofvarious plants have different relationships of the isozymes. The pIvalue of a protein can be determined by isoelectric focussing, which isknown to the skilled artisan.

The main isoform of the enzyme has an apparent molecular weight of 54kDa.

In particular, the preparation of the invention comprises isoformshaving pI values of 6.8, 7.1 and 7.6.

The invention also relates to a method of preparing “plantified”carbohydrate units of human and other vertebrate glycoproteins, whereinfucose units as well as GlcNAc-α1,3-fucosyl transferase encoded by anabove-described DNA molecule are admixed to a sample that comprises acarbohydrate unit or a glycoprotein, respectively, so that fucose inα1,3-position will be bound by the GlcNAc-α1,3-fucosyl transferase tothe carbohydrate unit or to the glycoprotein, respectively. By themethod according to the invention for cloning GlcNAc-α1,3-fucosyltransferase it is possible to produce large amounts of purified enzyme.To obtain a fully active transferase, suitable reaction conditions areprovided. It has been shown that the transferase has a particularly highactivity at a pH of approximately 7, if 2-(N-morpholino)-ethane sulfonicacid-HCl is used as the buffer. In the presence of bivalent cations, inparticular Mn²⁺, the activity of the recombinant transferase isenhanced. The carbohydrate unit is admixed to the sample either inunbound form or bound to a protein. The recombinant transferase isactive for both forms.

The invention will be explained in more detail by way of the followingexamples and drawing figures to which, of course, it shall not berestricted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a and 1 b show, as curves, the measured amounts of protein andthe measured enzyme activity in the individual fractions of the eluate;

FIG. 2 shows an electrophoresis gel analysis of GlcNAc-α1,3-fucosyltransferase;

FIG. 3 shows the result of the isoelectric focussing and the measuredtransferase activity of the individual isoforms;

FIG. 4 shows the N-terminal sequences of 4 tryptic peptides 1-4 as wellas the DNA sequence of three primers, S1, A2 and A3;

FIGS. 5 a and 5 b show the cDNA sequence of α1,3-fucosyl transferase;

FIGS. 6 a and 6 b show the amino acid sequence of α1,3-fucosyltransferase derived therefrom;

FIG. 7 is a schematic representation of the α1,3-fucosyl transferase aswell as the hydrophobicity of the amino acid residues;

FIG. 8 shows a comparison of the conserved motifs of various fucosyltransferases;

FIG. 9 shows a comparison of the fucosyl transferase activity of insectcells transfected with the α1,3-fucosyl transferase gene with that of anegative control;

FIGS. 10 a and 10 b show structures of different acceptors of theα1,3-fucosyl transferase;

FIGS. 11 and 12 show mass spectra; and

FIG. 13 shows the result of a HPLC.

EXAMPLES Example 1

Isolation of the Core-α1,3-Fucosyl Transferase

All the steps were carried out at 4° C. Mung bean seedlings werehomogenized in a mixer, 0.75 volumes of extraction buffer being used perkg of beans. Subsequently, the homogenate was filtered through twolayers of cotton fabric, and the filtrate was centrifuged for 40 min at30000×g. The supernatant was discarded, and the pellet was extractedwith solution buffer over night with continuous stirring. Subsequentcentrifugation at 30000×g for 40 min yielded the triton extract.

The triton extract was purified as follows:

Step 1: The triton extract was applied to a microgranular diethyl aminoethyl cellulose anion exchanger DE52 cellulose column (5×28 cm) fromWhatman, which previously had been calibrated with buffer A. Thenon-bound fraction was further treated in step 2.

Step 2: The sample was applied to an Affi-Gel Blue column (2, 5×32)column calibrated with buffer A. After washing of the column with thisbuffer, adsorbed protein was eluted with buffer A comprising 0.5 M NaCl.

Step 3: After dialysis of the eluate from step 2 against buffer B, itwas applied to an S-Sepharose column calibrated with the same buffer.Bound protein was eluted with a linear gradient of from 0 to 0.5 M NaClin buffer B. Fractions with GlcNAc-α1,3-fucosyl transferase were pooledand dialyzed against buffer C.

Step 4: The dialyzed sample was applied to a GnGn-Sepharose columncalibrated with buffer C. The bound protein was eluted with buffer Ccomprising 1 M NaCl instead of MnCl₂.

Step 5: Subsequently, the enzyme was dialyzed against buffer D andapplied to a GDP-Hexanolamine-Sepharose column. After having washed thecolumn with buffer D, the transferase was eluted by substituting MgCl₂and NaCl with 0.5 mM GDP. Active fractions were pooled, dialyzed against20 mM Tris-HCl buffer, pH 7.3, and lyophilized.

The enzymatic activity of the GlcNAc-α1,3-fucosyl transferase wasdetermined by using GnGn peptide and GDP-L-[U-¹⁴C]-fucose at substrateconcentrations of 0.5 and 0.25 each, in the presence of2-(N-morpholino)ethanesulfonic acid-HCl buffer, Triton X-100, MnCl₂,GlcNAc and AMP (according to Staudacher et al., 1998, Glycoconjugate J.15, 355-360; Staudacher et al., 1991, Eur. J. Biochem. 199, 745-751).

Protein concentrations were determined by aid of the bicinchoninic acidmethod (Pierce) or, in the final steps of enzyme purification, by meansof amino acid analysis (Altmann 1992, Anal. Biochem. 204, 215-219).

In FIGS. 1 a and 1 b, the measured amounts of protein and the measuredenzyme activity in the individual fractions of the eluate areillustrated as curves. FIG. 1 a shows the above-described separation onthe S-Sepharose column, FIG. 1 b shows the separation on theGnGn-Sepharose column, the circle representing protein, the black, fullcircle representing GlcNAc-α1,3-fucosyl transferase, and the squareillustrating N-acetyl-β-glucosaminidase. One U is defined as that amountof enzyme which transfers 1 mmol of fucose onto an acceptor per minute.

Table 1 shows the individual steps of transferase purification.

TABLE 1 Puri- Total Total Specific fication Purification proteinactivity activity factor Yield step mg mU mU/mg -fold % Triton X-10091500 4846 0.05 1 100 extract DE52 43700 4750 0.10 2 98.0 Affigel Blue180.5 4134 23 460 85.3 S-Sepharose 8.4 3251 390 7800 67.1 GnGn-Sepharose0.13¹ 1044 8030 160000 21.5 GDP-Hexanolamine- 0.02¹ 867 43350 86700017.9 Sepharose ¹determined by amino acid analysisExtraction buffer:

0.5 mM Dithiothreitol

1 M EDTA

0.5% Polyvinyl polypyrrolidone

0.25 M Sucrose

50 mM Tris-HCl buffer, pH 7.3

Solution buffer:

0.5 mM Dithiothreitol

1 M EDTA

0.5% Triton X-100

1 mM Tris-HCl, pH 7.3

Buffer A:

25 mM Tris-HCl buffer, pH 7.3, comprising:

0.1% Triton X-100 and

0.02% NaN₃

Buffer B:

25 mM Na citrate buffer, pH 5.3, comprising:

0.1% Triton X-100 and

0.02% NaN₃

Buffer C:

25 mM Tris-HCl buffer, pH 7.3, comprising:

5 mM MnCl₂nd

0.02% NaN₃

Buffer D:

25 mM Tris-HCl, pH 7.3, comprising:

10 mM MgCl₂

0.1 M NaCl, and

0.02% NaN₃

Example 2

SDS-PAGE and Isoelectric Focussing

An SDS-PAGE was carried out in a Biorad Mini-protean cell on gels with12.5% acrylamide and 1% bisacrylamide. The gels were stained either withCoomassie Brilliant Blue R-250 or Silver. Isoelectric focussing of thefucosyl transferase was carried out on prefabricated gels having a pIrange of between 6-9 (Servalyt precotes 6-9, Serva). The gels werestained with silver according to the producer's protocol. For thetwo-dimensional electrophoresis, lanes were cut out of the focussinggel, treated with S-alkylating reagents and SDS and subjected to anSDS-PAGE, as described above.

FIG. 2 shows the illustration of an electrophoresis gel ofGlcNAc-α1,3-fucosyl transferase, the two-dimensional electrophoresisbeing indicated on the left-hand side, and the one-dimensional SDS-PAGEbeing illustrated on the right-hand side. The lane denoted by A is astandard, the lane denoted by B is the GlcNAc-α1,3-fucosyl transferasefrom the GnGn-Sepharose column, and the lane denoted by C is the“purified” GlcNAc- α1,3-fucosyl transferase, i.e. the fraction of theGDP Hexanolamine Sepharose column. The two bands at 54 and 56 kDarepresent isoforms of the transferase.

FIG. 3 shows the result of the isoelectric focussing. Lane A was stainedwith silver, on lane B, the activity of the transferase isoforms wastested. The activity is indicated as % fucose which had been transferredfrom GDP-fucose onto the substrate.

Example 3

Peptide Sequencing

For sequencing of the protein, bands were cut out of theCoomassie-stained SDS-Polyacrylamide gel, carboxyamidomethylated andcleaved with trypsin according to Görg et al. 1988, Electrophoresis, 9,681-692. The tryptic peptides were separated with the reverse phase HPLCon a 1.0×250 mm Vydac C18 at 40° C. at a flow rate of 0.05 ml/min,wherein a HP 1100 apparatus (Hewlett-Packard) was used. The isolatedpeptides were separated with a Hewlett-Packard G1005 A ProteinSequencing System according to the producer's protocol. Furthermore, thepeptide mixture was analyzed by Ingel digestion with MALDI-TOF MS (seebelow).

FIG. 4 shows the N-terminal sequences of 4 tryptic peptides 1-4 (SEQ IDNO: 5-8). Departing from the first three peptides, primers S1, A2 and A3were prepared (SEQ ID NO: 9-11).

Example 4

RT-PCR and cDNA Cloning

The entire RNA was isolated from a 3-day-old mung bean hypocotyl,wherein the SV Total RNA Isolating System of Promega was used. Toprepare the first strand cDNA, the entire RNA was incubated for 1 h at48° C. with AMV reverse transcriptase and oligo(dT) primers, wherein theReverse Transcription System of Promega was used.

The first strand cDNA was subjected to a PCR, wherein a combination ofsense and antisense primers was used:

To 10 μl of the reverse transcription reaction mixture, the followingwas added:

50 μl with 0.1 mmol of each primer, 0.1 mM dNTPs, 2 mM MgCl₂, 10 mMTris-HCl buffer, pH 9.0, 50 mM KCl and 0.1% Triton X-100.

After a first denaturing step at 95° C. for 2 min, 40 cycles of 1 min at95° C., 1 min at 49° C. and 2 min at 72° C. were passed. The lastextension step was carried out at 72° C. for 8 min. PCR products weresubcloned into the pCR2.1 vector, with the TA Cloning Kit of Invitrogenbeing used, and sequenced. The products of this PCR were two DNAfragments with lengths of 744 bp and 780 bp, both DNA fragments havingthe same 5′-end (cf. also FIG. 7).

Starting from these two DNA fragments, the missing 5′ and 3′ regions ofthe CDNA were obtained by 5′ and 3′ rapid amplification of CDNA ends(RACE), wherein the RACE Kit of Gibco-BRL was used. As the antisenseprimer, the universal amplification primer of the kit, and as the senseprimer, either 5′-CTGGAACTGTCCCTGTGGTT-3′ (SEQ ID NO: 12) or5′-AGTGCACTAGAGGGCCAGAA-3′ (SEQ ID NO: 13) were used. As the senseprimer, also the shortened anchor primer of the kit, and as theantisense primer, 5′-TTCGAGCACCACAATTGGAAAT-3′ (SEQ ID NO: 14) or5′-GAATGCAAAGACGGCACGATGAAT-3′ (SEQ ID NO: 15) were used.

The PCR was carried out with an annealing temperature of 55° C. andunder the above-described conditions. The 5′ and 3′ RACE products weresubcloned into the pCR2.1 vector and sequenced: The sequences of thesubcloned fragments were sequenced by means of the didesoxynucleotidemethod (ABI PRISM Dye Terminator Cycle Sequencing Ready reaction Kit andABI PRISM 310 Genetic analyser (Perkin Elmer)). T7 and M13 forwardprimers were used for the sequencing of the products cloned into vectorpCR2.1. Both strands of the coding region were sequenced by the ViennaVBC Genomics-Sequencing Service, infrared-labelled primers (IRD700 andIRD800) and an LI-COR Long Read IR 4200 Sequencer (Lincoln, Nebr.) beingused.

FIGS. 5 a and 5 b show the entire cDNA which has a size of 2198 bp andan open reading frame of 1530 bp (SEQ ID NO: 1). The open reading frame(start codon at base pairs 211-213, stop codon at base pairs 1740-1743)codes for a protein of 510 amino acids having a molecular weight of 56.8kDA and a theoretical pI value of 7.51.

FIGS. 6 a and 6 b show the cDNA-derived amino acid sequence of theGlcNAc-α1,3-fucosyl transferase (SEQ ID NO: 2). Sites for theasparagine-bound glycosylation are at Asn346 and Asn429. In FIG. 7, theschematic GlcNAc-α1,3-fucosyl transferase-cDNA (top) and the derivedhydrophobicity index of the encoded protein (bottom) are illustrated, apositive hydrophobicity index meaning an increased hydrophobicity.Therebetween, the sizes of the two above-indicated PCR products areshown in relationship to the complete cDNA. The coding region isillustrated by the beam, “C” coding for the postulated cytoplasmaticregion, T for the postulated transmembrane region, and G for thepostulated Golgi lumen catalytic region of transferase. The analysis ofthe DNA sequence by “TMpred” (from EMBnet, Switzerland) gave an assumedtransmembrane region between Asn36 and Gly54. The C-terminal region ofthe enzyme probably comprises the catalytic region and consequentlyshould point into the lumen of the Golgi apparatus. According to this,this transferase seems to be a type II transmembrane protein like allthe hitherto analyzed glycosyl transferases which are involved inglycoprotein biosynthesis (Joziasse, 1992, Glycobiology 2, 271-277). Thegray regions represent the four tryptic peptides, the hexagons representthe potential N-glycosylation sites. A BLASTP search in all data banksaccesible via NCBI showed a similarity between the GlcNAc-α1,3-fucosyltransferase and other α1,3/4-fucosyl transferases, e.g. human fucosyltransferase VI. At 18-21% (examined by SIM-LALNVIEW, Expase,Switzerland), the total similarity was beyond any significance.Nevertheless, a sequence range of 35 amino acids (SEQ ID NO: 4) shows astrikingly high homology to other α1,3/4-fucosyl transferases (FIG. 8).This sequence region is located between Glu267 and Pro301 of SEQ ID NO:2.

Example 5

Expression of Recombinant GlcNAc-α1,3-Fucosyl Transferase in InsectCells

The encoding region of the assumed GlcNAc-α1,3-fucosyl transferaseincluding cytoplasmatic and transmembrane region was amplified with theforward primer 5′-CGGCGGATCCGCAATTGAATGATG-3′ (SEQ ID NO: 16) andreverse primer 5′-CCGGCTGCAGTACCATTTAGCGCAT-3′ (SEQ ID NO: 17) by meansof the Expand High Fidelity PCR System of Boehringer Mannheim. The PCRproduct was double-digested with PstI and BamHI and subcloned inalkaline phosphatase-treated baculovirus transfer vector pVL1393 whichpreviously had been digested with PstI and BamHI. To ensure a homologousrecombination, the transfer vector was co-transfected with Baculo Goldviral DNA (PharMingen, Sand Diego, Calif.) in Sf9 insect cells in IPL-41Medium with lipofectin. After an incubation of 5 days at 27° C., variousvolumes of the supernatant with the recombinant virus were used forinfecting the Sf21 insect cells. After an incubation of 4 days at 27° C.in IPL-41 Medium with 5% FCS, the Sf1 cells were harvested and washed 2×with phosphate-buffered saline solution. The cells were resuspended in25 mM Tris HCl buffer, pH 7.4, with 2% Triton X-100 and broken up bysonication on ice.

Example 6

Assay for GlcNAc-α1,3-Fucosyl Transferase Activity

The homogenate and the cell supernatant were assayed forGlcNAc-α1,3-fucosyl transferase. Blind samples were carried out withrecombinant baculovirus which codes for the tobacco-GlcNAc-transferase I(Strasser et al., 1999, Glycobiology, in the process of printing).

FIG. 9 shows the measured enzyme activity of the recombinantGlcNAc-α1,3-fucosyl transferase as well as of the negative control. Atbest, the enzyme activity of the cotransfected cells and theirsupernatant was 30× higher than that of the negative controls. Thisendogenous activity which is measurable in the absence of therecombinant transferase, substantially comes from theinsect-α1,6-fucosyl transferase and only a low percentage thereof comesfrom the GlcNAc-α1,3-fucosyl transferase. Accordingly, the increase inthe GlcNAc-α1,3-fucosyl transferase coming from the recombinantbaculoviruses is far more than the 100-fold.

The enzyme exhibited a broad maximum activity around a pH of 7.0, if theactivity was measured in 2-(N-morpholino)-ethanesulfonic acid-HClbuffer. As is apparent in Table 2, the addition of bivalent cations, inparticular Mn²⁺, enhances the activity of the recombinant transferase.

TABLE 2 Relative Activity Additive (Acceptor: GnGn-peptide) (conc. 10mM) % none 21 EDTA 18 MnCl₂ 100 CaCl₂ 82 MgCl₂ 52 CdCl₂ 44 CoCl₂ 35CuCl₂ 3 NiCl₂ 24 ZnCl₂ 0.6

Table 3 shows that among the acceptors used, the GnGn-peptide exhibitsthe highest incorporation rates under standard test conditions, followedclosely by GnGnF⁶eptide and M5Gn-Asn. A transfer to the MM peptide couldnot be found, which MM peptide does not comprise the reducing GlcNAc-endat the 3-bound mannose. This structure seems to be necessary for thecore fucosyl transferase. The recombinant transferase, moreover, wasinactive relative to the acceptors commonly used, the α3/4-fucosyltransferases used for determining the blood groups, which transfer thefucose to GlcNAc at the non-reducing ends of oligosaccharides. Theapparent K_(m)-values for the acceptor substrate GnGn peptide,GnGnF⁶peptide, M5Gn-Asn, and for the donor substrate GDP-fucose, wereassessed to be 0.19, 0.13, 0.23 and 0.11, respectively. The structuresof the molecules are illustrated in FIGS. 10 a and 10 b.

TABLE 3 Rel. Activity K_(m)-Value Acceptor Substrate % mM GnGn-peptide100 0.19 GnGnF⁶-peptide 87 0.13 M5Gn-Asn 71 0.23 MM-peptide 0Galβ-4GlcNAc 0 Galβ1-3GlcNAc 0 Galβ1-3GlcNAcβ1-3Galβ1-4Glc 0

Example 7

Mass Spectrometry of the Fucosyl Transferase Product

Dabsylated GnGn hexapeptide (2 nmol) was incubated with the insect cellhomogenate comprising the recombinant GlcNAc-α,3-fucosyl transferase(0.08 mU) in the presence of non-radioactive GDP-L-fucose (10 nmol), 2(N-morpholino)-ethanesulfonic acid-HCl buffer, Triton X-100, MnCl₂,GlcNAc and AMP. A negative control was carried out with a homogenate ofthe infected insect cells for the blind samples. The samples wereincubated for 16 h at 37° C. and analyzed by means of MALDI TOF massspectrometry.

Mass spectrometry was performed on a DYNAMO (Therrmo Bio-Analysis, SantaFe, N. Mex.), a MALDI-TOF MS which is capable of dynamic extraction(synonym for late extraction). Two types of sample matrix preparationswere used: peptides and dabsylated glycopeptides were dissolved in 5%formic acid, and aliquots were applied to the target, air-dried, andcovered with 1% α-cyano-4-hydroxy cinnamic acid. Pyridyl-aminatedglycans, reduced oligosaccharides and non-derivatized glycopeptides werediluted with water, applied to the target and air-dried. After additionof 2% 2.5-dihydroxy benzoic acid, the samples were immediately dried byapplying a vacuum.

FIG. 11 shows the mass spectrum of these samples, A being the negativecontrol: The main peak (S) shows theDabsyl-Val-Gly-Glu-(GlcNAc₄Man₃)Asn-Arg-Thr substrate, the calculated[M+H]⁺ value being 2262.3. This substrate also appears as sodiumaddition product and as smaller ion which has been formed byfragmentation of the Azo function of the Dabsyl group, at (S*). A smallproduct amount (P, [M+H]⁺=2408.4) is a consequence of the endogenousα1,6-fucosyl transferase. The peak at m/z=2424.0 shows the incompletede-galactosylation of the substrate. The mass spectrum B shows thesample with recombinant α1,3-fucosyl transferase. The main peak (P)represents the fucosylated product, (P*) its fragmented ion.

In addition, aliquots of both samples were mixed with each other so asto obtain similar concentrations of substrate and product (sample A).This mixture was diluted with 0.1 M ammonium acetate, pH 4.0, comprising10 mU of N-glycosidase A (sample B), or with 50 mM Tris/HCl, pH 8.5,comprising 100 mU (1 U hydrolyses 1 mmol of substrate per min) ofN-glycosidase F (sample C). After 2 and 20 h, small aliquots of thesemixtures were taken and analyzed by means of MALDI-TOF MS.

In FIG. 12, the three mass spectra of samples A, B and C areillustrated. The undigested sample A shows two main peaks: the substrateat 2261.4 m/z, and the fucosylated product at 2407.7 m/z. The middlecurve shows the mass spectrum of sample B, treated with N-glycosidase A,which hydrolyses both glycopeptides. The peak at 963.32 constitutes thedeglycosylated product. The lower curve shows the mass spectrum ofsample C. The N-glycosidase F is not able to hydrolyse α1,3-fucosylatedsubstrates, so that the spectrum has the peak at 2406.7 m/z of thefucosylated product, whereas the peak of the hydrolysed substrateappears at 963.08 m/z.

Example 8 HPLC-Analysis of the Pyridyl-Aminated Fucosyl TransferaseProduct

The two above-described samples (fucosylated product and negativecontrol) were digested with N-glycosidase A. The oligosaccharidesobtained were pyridyl-aminated and analysed by means of reverse phaseHPLC (Wilson et al., 1998, glycobiology 8, 651-661; Kubelka et al.,1994, Arch. Biochem. Giophys. 308, 148-157; Hase et al., 1984, J.Biochem. 95, 197-203).

In FIG. 13, the top diagram B represents the negative control, whereinin addition to the residual substrate (GnGn-peptide) α1,6-fucosylatedproduct is visible. A has a peak at a substantially shorter retentiontime, which is specific of reducing fucose bound to GlcNAc-α1,3.

In the bottom diagram, the isolated transferase product prior to (curveA) and following (curve B) digestion by N-acetyl-βglucosaminidase wascompared with MMF³ honeybee phospholipase A₂ (curve C).

1. A recombinant glycoprotein comprising: (a) a peptide sequencecomprising less than 50% of the α1,3-bound fucose residues occurring inrecombinant glycoproteins expressed in a plant or plant cell expressionsystem in which the endogenous GlcNAc-α1,3-fucosyltransferase productionis not suppressed or completely blocked and (b) a non-reducing GlcNAcend, wherein said recombinant glycoprotein is expressed in a plant orplant cell expression system in which the endogenousGlcNAc-α1,3-fucosyltransferase production is suppressed or completelyblocked.
 2. The recombinant qlycoprotein according to claim 1, whereinthe glycoprotein is a human protein.
 3. The recombinant glycoproteinaccording to claim 1 or 2, wherein the peptide sequence comprises lessthan 20% of the α1,3-bound fucose residues occurring in recombinantglycoproteins expressed in a plant or plant cell expression system inwhich the endogenous GlcNAc-α1,3-fucosyltransferase production is notsuppressed or completely blocked.
 4. The recombinant glycoproteinaccording to claim 3, wherein the peptide sequence comprises 0% of theα1,3-bound fucose residues occurring in recombinant glycoproteinsexpressed in a plant or plant cell expression system in which theendogenous GlcNAc-α1,3-fucosyltransferase production is not suppressedor completely blocked.
 5. A method for preparing a recombinantglycoprotein according to claim 1 or 2, comprising expressing theglycoprotein in a plant or plant cell expression system, in which anendogenous GlcNAc-α1,3-fucosyltransferase production has been suppressedby at least 50%.
 6. The method according to claim 5, wherein theendogenous GlcNAc-α1,3-fucosyltransferase production has been completelyblocked.
 7. The method according to claim 5, wherein the suppression iseffected by a knock-out mutation or by antisense RNA inhibition.
 8. Themethod according to claim 7, wherein the suppression is effected byantisense RNA inhibition.
 9. The method according to claim 7, whereinthe suppression is effected by a knock-out mutation.
 10. The methodaccording to claim 8, wherein the RNA inhibition comprises using apolynucleotide comprising a sequence selected from the group consistingof: (a) base pair 211 to base pair 1740 of SEQ ID NO. 1; (b) a sequencehaving at least 50% identity with base pair 211 to base pair 1740 of SEQID NO. 1; (c) a sequence which hybridizes with base pair 211 to basepair 1740 of SEQ ID NO. 1 under stringent conditions; and (d) a sequencethat has degenerated to base pair 211 to base pair 1740 of SEQ ID NO. 1as a consequence of the genetic code.
 11. The method according to claim9, wherein the knock-out mutation comprises a deletion, insertion orsubstitution mutation in a sequence coding for a protein having afucosyltransferase activity, and wherein said deletion, insertion orsubstitution mutation is created by using an expression systemcomprising a polynucleotide comprising a sequence selected from thegroup consisting of: (a) base pair 211 to base pair 1740 of SEQ ID NO.1; (b) a sequence having at least 50% identity with base pair 211 tobase pair 1740 of SEQ ID NO. 1; (c) a sequence which hybridizes withbase pair 211 to base pair 1740 of SEQ ID NO. 1 under stringentconditions; and (d) a sequence that has degenerated to base pair 211 tobase pair 1740 of SEQ ID NO. 1 as a consequence of the genetic code.